This application is for competitive renewal of an R01 project focused on defining determinants of bacterial persistence in biofilms during otitis media infections. Otitis media is caused by airway opportunists such as Haemophilus influenzae (Hi, which forms biofilm communities that promote bacterial persistence within the middle-ear chamber. During the first four years of support we have defined mechanism(s) involved in biofilm formation by Hi, and proved that biofilms promote bacterial persistence in vivo. For the next project period we will define determinants of biofilm resistance to host clearance. We hypothesize that biofilms promote bacterial resistance to clearance because of activation of bacterial stress-responses within the biofilm, and inherent resistance to host phagocytes. In order to address these hypotheses we will complete the following Specific Aims: Specific Aim 1: To delineate role(s) of bacterial stress-responses in Hi persistence during experimental otitis media. Specific Aim 2: To define resistance of Hi biofilms to phagocytic and extracellular killing. Although it is clear that the biofilm mode of growth is involved in the majority of persistent infections, much remains to be learned about the determinants of bacterial resistance to clearance within biofilms. The completion of the proposed work will enhance our understanding of the role of biofilms within the clinical context of otitis media. PUBLIC HEALTH RELEVANCE: Otitis media is a major common and costly pediatric illness worldwide, accounting for billions of dollars per year in total economic impact. Otitis media infections are the leading reason for pediatric office visits, new antibiotic prescriptions, and surgical instillation of tympanic drain tubes to relieve chronic and recurrent otitis media is the most commonly performed surgical procedure in the U.S. Haemophilus influenzae has long been recognized as the most common bacterial causes of otitis media, and it is now clear that H. influenzae forms biofilms that are important determinants of persistent infections. The focus of this application is to understand how these biofilm communities promote bacterial persistence in vivo. Understanding how bacteria form these biofilms will be an important step in learning to better diagnose, prevent, and/or treat chronic infections.